Cell Cycle and Apoptosis October 12, 2017

Classes of Bcl2 Proteins Bcl2 proteins –regulate apoptosis through controlling the release of cytochrome c

Pro-Apoptotic BH123

Regulation of Intrinsic Pathway BH3-only proteins activate apoptosis through direct binding with anti-apoptotic proteins -p53 activates BH3-only proteins (Puma and Noxa) -Bid – extrinsic and intrinsic pathways

BNIP3 Family -BH3-only protein, pro-apoptotic Family memebers: BNIP3 (Bcl-2/EiB-19K interacting protein 3) BNIP3L, B5, BNIP3α, NIX, BNIP3h

Regulation of BNIP3 family -Up regulated in hypoxic conditions, 2-fold -HIF-1α activates BNIP3 -PLAGL2 can also activate BNIP3

Regulation of BNIP3 family How does Apoptosis lead to Cancer?

Regulation of BNIP3 family How does Apoptosis lead to Cancer? Hypoxic stress in the tumor microenvironment contributes to the evolution of malignant tumor cells with increased invasive and angiogenic potentials Correlation between tumor hypoxia and increased metastatic efficiency and resistance to radiation and chemotherapy Conflicting signals between: Death factors Vs. Survival factors BNIP3 VEGF, FGF, IGF

Regulation of BNIP3 family Inactivation or Down regulation of BNIP3

Regulation of BNIP3 family Pancreatic Cancers -Hypermethylation of BNIPs promoter -Other hypoxia induced genes were up regulated Colorectal Cancers -histone deacetylation

Extrinsic Pathway DISC – death-inducing signaling complex Inhibitors such as decoy receptors and intracellular blocking proteins

TRAIL Extrinsic Apoptotic Pathway

TRAIL Tumor necrosis Factor-related apoptosis-inducing ligand (TRAIL) -Induction of Extrinsic Apoptotic pathway -Many cancers are sensitive to TRAIL-induced apoptosis -Some are not: Pancreatic cancer, melanoma, and neuroblastoma

TRAIL Receptors 5 Receptors -DR4 and DR5, have the Death Domain to trigger apoptosis -DcR1 and DcR2, Decoy Receptors, cannot trigger apoptosis -OPG, secreted and detected in circulation DR and DcR are both widely expressed in many human cells

Single Nucleotide Polymorphisms SNPs -a DNA nucleotide variation occurring in the genome between two members of the same species

Occurrance in Humans -1 in 1000 bases, 0.1% -3 to 10 million SNPs in the human genome

Importance of SNPs

Cancer Susceptibility

Polymorphisms in DR4 Receptors Ovarian and Bladder Cancer Cell lines -A to G change in DR4 -resulting in K441R mutation, Lysine to Arginine -Less effective in cell killing

Polymorphisms in DR4 Receptors Lung, head and neck, and gastric adenocarinoma -C to G change in DR4 -resulting in T209R, Threonine to Arginine -G to A change in DR4 -resulting in R141H, Arginine to Histidine -Abnormal death receptor trimerization Homozygous for both T209R and R141H -13% Normal individuals -35% non-small cell lung cancer -47% primary head and neck cacner -44% gastric adenocarcinomas

Mutations in DR4 Receptors Breast Cancer specimens -34 specimens which metastasized -3 mutations in DR4 -single base changes in the death domain -Also showed an increase in LOH -23 specimens had not metastasized -0 mutations in DR4

Mutations in DR5 Receptors Primary head and neck cancer -40 specimens -2 mutations in DR5 -A 2 base insertion in death domain -Truncated protein Non-small cell lunch cancer -104 specimens -11 mutations in the death domain of DR5 -7 of the 11 showed LOH

Mutations in DR5 Receptors Breast Cancer specimens -34 specimens which metastasized -4 mutations in DR4 -2 were in the death domain, 2 adjacent -Also showed an increase in LOH -23 specimens had not metastasized -0 mutations in DR4 Placement of these mutations in other cells resulted in suppression of apoptosis

TRAIL Resistance: Caspase-8 -Down regulation of Caspase-8 in TRAIL Resistant Cancers -Gene deletion (rare) -Promoter methylation - Treatment of cells with demethylating agent restored apoptosis Caspase-8 importance -Caspase-8 Inhibitor, Z-IETD-FMK halted apoptosis -Caspace-10 was not a functional substitute X

TRAIL Resistance: Bcl-2 family -Up regulation of Bcl-2 or Bcl-XL -Pancreatic adenocarcinoma -Bcl-XL was up regulated in 3 cell lines Neuroblastoma, glioblastoma, and breast cancer -Bcl-2 was up regulated in cell lines X

TRAIL Resistance: Bax and Bak -Bax and Bak are pro-apoptotic genes -They have redundant functions as seen in Bax -/- or Bak -/- mice -To result in TRAIL-induced apoptosis, the mice must be both Bax -/- Bak -/- -They would need to be down regulated X

TRAIL Resistance: Bax and Bak -Bax and Bak are pro-apoptotic genes -They have redundant functions as seen in Bax -/- or Bak -/- mice -To result in TRAIL-induced apoptosis, the mice must be both Bax -/- Bak -/- -They would need to be down regulated X

TRAIL Resistance: IAPs and anti-IAPs -IAPs have domains called baculovirus inhibitory repeat (BIR) thought to interact with different caspases -Trail Resistance by: -Up regulation of IAPs -Down regulation of anti-IAPs X X

TRAIL Resistance: IAPs

TRAIL Resistance: NF-κB Controversial

TRAIL-Induced Apoptosis